Novel human hydroxylases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in industrial, therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/294,076, which was filed on May 29, 2001, and isherein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding proteinssharing sequence similarity with mammalian hydroxylases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded protein, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosedpolynucleotides, antagonists and agonists of the proteins, and othercompounds that modulate the expression or activity of the proteinsencoded by the disclosed polynucleotides, which can be used fordiagnosis, drug screening, clinical trial monitoring, the treatment ofdiseases and disorders, and cosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

[0003] Hydroxylases are enzymes that mediate the cleavage of hydroxylgroups, and many hydroxylases act in biological pathways suitable fordrug intervention.

3. SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel human proteins,and the corresponding amino acid sequences of these proteins. The novelhuman proteins (NHPs) described for the first time herein sharestructural similarity with animal hydroxylases, and particularlytryptophan hydroxylases, which are involved in a rate-limiting step inthe biosynthesis of a number of neurologically active compounds,including, but not limited to, DOPA, serotonin and melatonin. Given thewell known physiological functions of the above compounds, the describedhydroxylases can be used to identify and/or develop agents useful formodulating behavior (i.e., treating anxiety, depression, hyperactivity,sleep disorders, etc.). The novel human nucleic acid (cDNA) sequencesdescribed herein encode proteins/open reading frames (ORFs) of 490, 486,485, and 484 amino acids in length (SEQ ID NOS:2, 4, 6, and 8,respectively).

[0005] In addition to the direct therapeutic use of the described NHPs,the invention also encompasses agonists and antagonists of the describedNHPs, including small molecules, large molecules, mutant NHPs, orportions thereof, that compete with native NHPs, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stem cell(“ES cell”) lines that contain gene trap mutations in a murine homologof at least one of the described NHPs. When the unique NHP sequencesdescribed in SEQ ID NOS:1-8 are “knocked-out” they provide a method ofidentifying phenotypic expression of the particular gene, as well as amethod of assigning function to previously unknown genes. In addition,animals in which the unique NHP sequences described in SEQ ID NOS:1-8are “knocked-out” provide a unique source in which to elicit antibodiesto homologous and orthologous proteins, which would have been previouslyviewed by the immune system as “self” and therefore would have failed toelicit significant antibody responses.

[0006] Additionally, the unique NHP sequences described in SEQ IDNOS:1-8 are useful for the identification of protein coding sequences,and mapping a unique gene to a particular chromosome. These sequencesidentify biologically verified exon splice junctions, as opposed tosplice junctions that may have been bioinformatically predicted fromgenomic sequence alone. The sequences of the present invention are alsouseful as additional DNA markers for restriction fragment lengthpolymorphism (RFLP) analysis, and in forensic biology, particularlygiven the presence of a nucleotide polymorphism within the describedsequences.

[0007] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists of, NHP expression and/or NHP activity that utilize purifiedpreparations of the described NHPs and/or NHP products, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0008] The Sequence Listing provides the sequences of the ORFs encodingthe described NHP amino acid sequences.

5. DETAILED DESCRIPTION OF THE INVENTION

[0009] The NHPs described for the first time herein are novel proteinsthat can be expressed in, inter alia, human cell lines, human fetalbrain, spinal cord, lymph node, prostate, testis, thyroid, pancreas,pericardium, hypothalamus, fetal kidney, fetal lung, 6 and 9-week oldembryos, osteosarcoma, and embryonic carcinoma cells.

[0010] The present invention encompasses the nucleotides presented inthe Sequence Listing, host cells expressing such nucleotides, theexpression products of such nucleotides, and: (a) nucleotides thatencode mammalian homologs of the described polynucleotides, includingthe specifically described NHPs, and the NHP products; (b) nucleotidesthat encode one or more portions of a NHP that correspond to functionaldomains of a NHP, and the polypeptide products specified by suchnucleotide sequences, including, but not limited to, the novel regionsof any active domain(s); (c) isolated nucleotides that encode mutantversions, engineered or naturally occurring, of a described NHP, inwhich all or a part of at least one domain is deleted or altered, andthe polypeptide products specified by such nucleotide sequences,including, but not limited to, soluble proteins and peptides; (d)nucleotides that encode chimeric fusion proteins containing all or aportion of a coding region of a NHP, or one of its domains (e.g., areceptor or ligand binding domain, accessory protein/self-associationdomain, etc.) fused to another peptide or polypeptide; or (e)therapeutic or diagnostic derivatives of the described polynucleotides,such as oligonucleotides, antisense polynucleotides, ribozymes, dsRNA,or gene therapy constructs, comprising a sequence first disclosed in theSequence Listing.

[0011] As discussed above, the present invention includes the human DNAsequences presented in the Sequence Listing (and vectors comprising thesame), and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1× SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., N.Y., at p. 2.10.3) andencodes a functionally equivalent expression product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of a DNA sequence that encodes and expresses an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2× SSC/0.1% SDS at 42° C. (Ausubel etal., 1989, supra), yet still encode a functionally equivalent NHPproduct. Functional equivalents of a NHP include naturally occurringNHPs present in other species, and mutant NHPs, whether naturallyoccurring or engineered (by site directed mutagenesis, gene shuffling,directed evolution as described in, for example, U.S. Pat. Nos.5,837,458 and 5,723,323 both of which are herein incorporated byreference in their entirety). The invention also includes degeneratenucleic acid variants of the disclosed NHP polynucleotide sequences.

[0012] Additionally contemplated are polynucleotides encoding a NHP ORF,or its functional equivalent, encoded by a polynucleotide sequence thatis about 99, 95, 90, or about 85 percent similar or identical tocorresponding regions of the nucleotide sequences of the SequenceListing (as measured by BLAST sequence comparison analysis using, forexample, the GCG sequence analysis package, as described herein, usingstandard default settings).

[0013] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHP nucleotide sequences. Such hybridization conditionsmay be highly stringent or less highly stringent, as described herein.In instances where the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules are generally about 16 to about 100 baseslong, or about 20 to about 80 bases long, or about 34 to about 45 baseslong, or any variation or combination of sizes represented therein thatincorporate a contiguous region of sequence first disclosed in theSequence Listing. Such oligonucleotides can be used in conjunction withthe polymerase chain reaction (PCR) to screen libraries, isolate clones,and prepare cloning and sequencing templates, etc.

[0014] Alternatively, such NHP oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a microarray or high-throughput“chip” format). Additionally, a series of NHP oligonucleotide sequences,or the complements thereof, can be used to represent all or a portion ofthe described NHP sequences. An oligonucleotide or polynucleotidesequence first disclosed in at least a portion of one or more of thesequences of SEQ ID NOS:1-8 can be used as a hybridization probe inconjunction with a solid support matrix/substrate (resins, beads,membranes, plastics, polymers, metal or metallized substrates,crystalline or polycrystalline substrates, etc.). Of particular note arespatially addressable arrays (i.e., gene chips, microtiter plates, etc.)of oligonucleotides and polynucleotides, or corresponding oligopeptidesand polypeptides, wherein at least one of the biopolymers present on thespatially addressable array comprises an oligonucleotide orpolynucleotide sequence first disclosed in at least one of the sequencesof SEQ ID NOS:1-8, or an amino acid sequence encoded thereby. Methodsfor attaching biopolymers to, or synthesizing biopolymers on, solidsupport matrices, and conducting binding studies thereon, are disclosedin, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305,4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405,the disclosures of which are herein incorporated by reference in theirentirety.

[0015] Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-8 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is usually within a range of between about 8to about 2000 nucleotides. Preferably the probes consist of 60nucleotides, and more preferably 25 nucleotides, from the sequencesfirst disclosed in SEQ ID NOS:1-8.

[0016] For example, a series of such NHP oligonucleotide sequences, orthe complements thereof, can be used in chip format to represent all ora portion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length, can partially overlap each other, and/orthe sequence may be represented using oligonucleotides that do notoverlap. Accordingly, the described polynucleotide sequences shalltypically comprise at least about two or three distinct oligonucleotidesequences of at least about 8 nucleotides in length that are each firstdisclosed in the described Sequence Listing. Such oligonucleotidesequences can begin at any nucleotide present within a sequence in theSequence Listing, and proceed in either a sense (5′-to-3′) orientationvis-a-vis the described sequence or in an antisense orientation.

[0017] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions, andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-8 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel components,or gene functions that manifest themselves as novel phenotypes.

[0018] Probes consisting of sequences first disclosed in SEQ ID NOS:1-8can also be used in the identification, selection, and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets, andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the intended target of thedrug. These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0019] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-8 can be utilized in microarrays, or other assay formats, toscreen collections of genetic material from patients who have aparticular medical condition. These investigations can also be carriedout using the sequences first disclosed in SEQ ID NOS:1-8 in silico, andby comparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

[0020] Thus the sequences first disclosed in SEQ ID NOS:1-8 can be usedto identify mutations associated with a particular disease, and also indiagnostic or prognostic assays.

[0021] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence, inconjunction with the presence of one or more specific oligonucleotidesequence(s) first disclosed in SEQ ID NOS:1-8. Alternatively, arestriction map specifying the relative positions of restrictionendonuclease digestion sites, or various palindromic or other specificoligonucleotide sequences, can be used to structurally describe a givensequence. Such restriction maps, which are typically generated by widelyavailable computer programs (e.g., the University of Wisconsin GCGsequence analysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor,Mich., etc.), can optionally be used in conjunction with one or morediscrete nucleotide sequence(s) present in the sequence that can bedescribed by the relative position of the sequence relative to one ormore additional sequence(s) or one or more restriction sites present inthe disclosed sequence.

[0022] For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6× SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP antisense molecules, useful, for example, inNHP gene regulation and/or as antisense primers in amplificationreactions of NHP nucleic acid sequences. With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences may be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

[0023] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0024] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0025] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0026] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

[0027] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.USA 85:7448-7451), etc.

[0028] Low stringency conditions are well-known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (and periodic updates thereof), and Ausubel et al.,1989, supra.

[0029] Alternatively, suitably labeled NHP nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0030] For example, the present sequences can be used in restrictionfragment length polymorphism (RFLP) analysis to identify specificindividuals. In this technique, an individual's genomic DNA is digestedwith one or more restriction enzymes, and probed on a Southern blot toyield unique bands for identification (as generally described in U.S.Pat. No. 5,272,057, incorporated herein by reference). In addition, thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e., another DNA sequence that is unique to a particularindividual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments.

[0031] Further, a NHP homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHP products disclosed herein. The template for thereaction may be genomic DNA, or total RNA, mRNA, and/or cDNA obtained byreverse transcription of mRNA prepared from human or non-human celllines or tissue known to express, or suspected of expressing, an alleleof a NHP gene.

[0032] The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0033] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown to express, or suspected of expressing, a NHP gene, such as, forexample, testis tissue). A reverse transcription (RT) reaction can beperformed on the RNA using an oligonucleotide primer specific for themost 5′ end of the amplified fragment for the priming of first strandsynthesis. The resulting RNA/DNA hybrid may then be “tailed” using astandard terminal transferase reaction, the hybrid may be digested withRNase H, and second strand synthesis may then be primed with acomplementary primer. Thus, cDNA sequences upstream of the amplifiedfragment can be isolated. For a review of cloning strategies that can beused, see, e.g., Sambrook et al., 1989, supra.

[0034] A cDNA encoding a mutant NHP sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known to express, or suspected of expressing, a NHP, in anindividual putatively carrying a mutant NHP allele, and by extending thenew strand with reverse transcriptase. The second strand of the cDNA isthen synthesized using an oligonucleotide that hybridizes specificallyto the 5′ end of the normal sequence. Using these two primers, theproduct is then amplified via PCR, optionally cloned into a suitablevector, and subjected to DNA sequence analysis through methodswell-known to those of skill in the art. By comparing the DNA sequenceof the mutant NHP allele to that of a corresponding normal NHP allele,the mutation(s) responsible for the loss or alteration of function ofthe mutant NHP gene product can be ascertained.

[0035] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of carrying, or known to carry, amutant NHP allele (e.g., a person manifesting a NHP-associated phenotypesuch as, for example, neurological disorders, such as, but not limitedto, depression, anxiety, Alzheimer's disease or Parkinson's disease,obesity, high blood pressure, connective tissue disorders, infertility,developmental disorders, etc.), or a cDNA library can be constructedusing RNA from a tissue known to express, or suspected of expressing, amutant NHP allele. A normal NHP gene, or any suitable fragment thereof,can then be labeled and used as a probe to identify the correspondingmutant NHP allele in such libraries. Clones containing mutant NHPsequences can then be purified and subjected to sequence analysisaccording to methods well-known to those skilled in the art.

[0036] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known toexpress, or suspected of expressing, a mutant NHP allele in anindividual suspected of carrying, or known to carry, such a mutantallele. In this manner, gene products made by the putatively mutanttissue can be expressed and screened using standard antibody screeningtechniques in conjunction with antibodies raised against a normal NHPproduct, as described below (for screening techniques, see, for example,Harlow and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.).

[0037] Additionally, screening can be accomplished by screening withlabeled NHP fusion proteins, such as, for example, alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In caseswhere a NHP mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to a NHP are likely to cross-react with acorresponding mutant NHP expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well-known in theart.

[0038] The invention also encompasses: (a) DNA vectors that contain anyof the foregoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators, and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include, but are not limited to, the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40 oradenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

[0039] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of a NHP, as well as compounds or nucleotide constructs thatinhibit expression of a NHP sequence (transcription factor inhibitors,antisense and ribozyme molecules, or open reading frame sequence orregulatory sequence replacement constructs), or promote the expressionof a NHP (e.g., expression constructs in which NHP coding sequences areoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

[0040] The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant NHPs, or inappropriately expressed NHPs, for thediagnosis of disease. The NHP proteins or peptides, NHP fusion proteins,NHP nucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of a NHPin the body. The use of engineered host cells and/or animals may offeran advantage in that such systems allow not only for the identificationof compounds that bind to the endogenous receptor for a NHP, but canalso identify compounds that trigger NHP-mediated activities orpathways.

[0041] Finally, the NHP products can be used as therapeutics. Forexample, soluble derivatives such as NHP peptides/domains correspondingto a NHP, NHP fusion protein products (especially NHP-Ig fusionproteins, i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP-mediated pathway), can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of a soluble NHP (or, for example, a suitablyderivatized NHP, e.g., with polyethylene glycol (PEG), albumin, etc.), aNHP-IgFc fusion protein, or an anti-idiotypic antibody (or its Fab) thatmimics the NHP, could activate or effectively antagonize the endogenousNHP receptor. Nucleotide constructs encoding such NHP products can beused to genetically engineer host cells to express such products invivo; these genetically engineered cells function as “bioreactors” inthe body delivering a continuous supply of a NHP, a NHP peptide, or aNHP fusion protein to the body. Nucleotide constructs encodingfunctional NHPs, mutant NHPs, as well as antisense and ribozymemolecules, can also be used in “gene therapy” approaches for themodulation of NHP expression. Thus, the invention also encompassespharmaceutical formulations and methods for treating biologicaldisorders.

[0042] Various aspects of the invention are described in greater detailin the subsections below.

5.1 THE NHP SEQUENCES

[0043] The cDNA sequences and the corresponding deduced amino acidsequences of the described NHPs are presented in the Sequence Listing.The described sequences were compiled from human gene trapped sequences,and cDNAs made from human fetal brain, fetal lung, and lymph node mRNAs(Edge Biosystems, Gaithersburg, Md.). As biologically validated (splicedand polyadenylated sequences), the described sequences are useful formapping the corresponding coding region of the human genome andparticularly for mapping exon splice junctions. The described NHPs areapparently encoded on human chromosome 12 (see GENBANK accession no.AC023966).

[0044] An A/T polymorphism was detected during the sequencing of theNHPs at the nucleotide position represented by, for example, position1125 of SEQ ID NO:1 (and the corresponding location in the other NHPnucleotide sequences), both of which result in an ala at the regioncorresponding to amino acid (aa) position 375 of, for example, SEQ IDNO:2 (and the corresponding location in the other NHP amino acidsequences). As these polymorphisms are coding single nucleotidepolymorphisms (SNPs), they are particularly useful in forensic analysis.

[0045] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721 and 5,837,458, which are herein incorporated byreference in their entirety.

[0046] NHP gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, worms, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees, may be usedto generate NHP transgenic animals.

[0047] Any technique known in the art may be used to introduce a NHPtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to, pronuclearmicroinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191);retrovirus-mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171-229, which is incorporated byreference herein in its entirety.

[0048] The present invention provides for transgenic animals that carrya NHP transgene in all their cells, as well as animals that carry atransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. A transgene may be integrated as asingle transgene, or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. A transgene may also be selectively introducedinto and activated in a particular cell-type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell-type ofinterest, and will be apparent to those of skill in the art.

[0049] When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

[0050] The transgene can also be selectively introduced into aparticular cell-type, thus inactivating the endogenous NHP gene in onlythat cell-type, by following, for example, the teaching of Gu et al.,1994, Science 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

[0051] Once transgenic animals have been generated, the expression ofthe recombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHP gene-expressing tissue may also beevaluated immunocytochemically using antibodies specific for the NHPtransgene product.

[0052] The present invention also provides for “knock-in” animals.Knock-in animals are those in which a polynucleotide sequence (i.e., agene or a cDNA) that the animal does not naturally have in its genome isinserted in such a way that it is expressed. Examples include, but arenot limited to, a human gene or cDNA used to replace its murine orthologin the mouse, a murine cDNA used to replace the murine gene in themouse, and a human gene or cDNA or murine cDNA that is tagged with areporter construct used to replace the murine ortholog or gene in themouse. Such replacements can occur at the locus of the murine orthologor gene, or at another specific site. Such knock-in animals are usefulfor the in vivo study, testing and validation of, intra alia, human drugtargets, as well as for compounds that are directed at the same, andtherapeutic proteins.

5.2 NHPS AND NHP POLYPEPTIDES

[0053] NHPs, NHP polypeptides, NHP peptide fragments, mutated,truncated, or deleted forms of the NHPS, and/or NHP fusion proteins canbe prepared for a variety of uses. These uses include, but are notlimited to, the generation of antibodies, as therapeutic products(particularly in the treatment of behavioral disorders such as, but notlimited to, dementia, insomnia, depression, anorexia, and premenstrualsyndrome, etc., as well as cancers such as, but not limited to,lymphoma, leukemia, sarcomas, carcinomas, myelomas, etc.), as reagentsin diagnostic assays, for the identification of other cellular geneproducts related to a NHP, as reagents in assays for screening forcompounds that can be used as pharmaceutical reagents useful in thetherapeutic treatment of mental, biological, or medical disorders anddisease. Given the similarity information and expression data, thedescribed NHPs can be targeted (by drugs, oligos, antibodies, etc.) inorder to treat disease, or to therapeutically augment the efficacy oftherapeutic or chemotherapeutic agents. In addition, the described NHPscan be used in drug screening assays similar to those described in, forexample, U.S. Pat. No. 6,048,850, herein incorporated by reference, inorder to identify compounds for treating diseases such as, for example,depression, anxiety, immune disorders, Alzheimer's disease, epilepsy,and Parkinson's disease.

[0054] The Sequence Listing discloses the amino acid sequences encodedby the described NHP polynucleotides. The NHPs display initiatormethionines in DNA sequence contexts consistent with translationinitiation sites, and a N-terminal signal sequence characteristic ofsecreted or membrane proteins (the signal sequence is often cleaved fromthe mature form of the protein incident to the protein translocatingacross the membrane).

[0055] The NHP amino acid sequences of the invention include the aminoacid sequences presented in the Sequence Listing, as well as analoguesand derivatives thereof, as well as any oligopeptide sequence of atleast about 10-40 amino acids, about 12-35 amino acids, or about 16-30amino acids in length first disclosed in the Sequence Listing. Further,corresponding NHP homologues from other species are encompassed by theinvention. In fact, any NHP encoded by the NHP nucleotide sequencesdescribed herein are within the scope of the invention, as are any novelpolynucleotide sequences encoding all or any novel portion of an aminoacid sequence presented in the Sequence Listing. The degenerate natureof the genetic code is well-known, and, accordingly, each amino acidpresented in the Sequence Listing is generically representative of thewell-known nucleic acid “triplet” codon, or in many cases codons, thatcan encode the amino acid. As such, as contemplated herein, the aminoacid sequences presented in the Sequence Listing, when taken togetherwith the genetic code (see, for example, Table 4-1 at page 109 of“Molecular Cell Biology”, 1986, J. Darnell et al., eds., ScientificAmerican Books, New York, N.Y., herein incorporated by reference), aregenerically representative of all the various permutations andcombinations of nucleic acid sequences that can encode such amino acidsequences.

[0056] The invention also encompasses proteins that are functionallyequivalent to the NHPs encoded by the presently described nucleotidesequences, as judged by any of a number of criteria, including, but notlimited to, the ability to bind and cleave a substrate of a NHP, theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,tyrosine phosphorylation, etc.). Such functionally equivalent NHPproteins include, but are not limited to, additions or substitutions ofamino acid residues within the amino acid sequence encoded by the NHPnucleotide sequences described herein, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions can be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0057] A variety of host-expression vector systems can be used toexpress the NHP nucleotide sequences of the invention. Where, as in thepresent instance, the NHP products or NHP polypeptides can be producedin soluble or secreted forms (by removing one or more transmembranedomains where applicable), the peptide or polypeptide can be recoveredfrom the culture media. Such expression systems also encompassengineered host cells that express a NHP, or a functional equivalent, insitu. Purification or enrichment of a NHP from such expression systemscan be accomplished using appropriate detergents and lipid micelles andmethods well-known to those skilled in the art. However, such engineeredhost cells themselves may be used in situations where it is importantnot only to retain the structural and functional characteristics of aNHP, but to assess biological activity, e.g., in certain drug screeningassays.

[0058] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining NHP nucleotide sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing NHPnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0059] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing a NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in-frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouyeand Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke andSchuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEXvectors (Pharmacia or American Type Culture Collection) can also be usedto express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

[0060] In an exemplary insect system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreignpolynucleotide sequences. The virus grows in Spodoptera frugiperdacells. A NHP coding sequence can be cloned individually into anon-essential region (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of a NHP coding sequence will result ininactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted sequence isexpressed (e.g., see Smith et al., 1983, J. Virol. 46:584; Smith, U.S.Pat. No. 4,215,051).

[0061] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NHP nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericsequence may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NHP product in infected hosts(e.g., see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHP nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHP gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHP coding sequenceis inserted, exogenous translational control signals, including,perhaps, the ATG initiation codon, may be provided. Furthermore, theinitiation codon should be in phase with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons canbe of a variety of origins, both natural and synthetic. The efficiencyof expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBitter et al., 1987, Methods in Enzymol. 153:516-544).

[0062] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the desired modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for the desired processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

[0063] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the NHP sequences described herein can be engineered. Ratherthan using expression vectors that contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then switched to a selective media. The selectable marker inthe recombinant plasmid confers resistance to the selection and allowscells to stably integrate the plasmid into their chromosomes and grow toform foci, which in turn can be cloned and expanded into cell lines.This method may advantageously be used to engineer cell lines thatexpress a NHP product. Such engineered cell lines may be particularlyuseful in screening and evaluation of compounds that affect theendogenous activity of a NHP product.

[0064] A number of selection systems may be used, including, but notlimited to, the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817)genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567;O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, whichconfers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc.Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre etal., 1984, Gene 30:147).

[0065] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.Another exemplary system allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system,the sequence of interest is subcloned into a vaccinia recombinationplasmid such that the sequence's open reading frame is translationallyfused to an amino-terminal tag consisting of six histidine residues.Extracts from cells infected with recombinant vaccinia virus are loadedonto Ni²⁺.nitriloacetic acid-agarose columns, and histidine-taggedproteins are selectively eluted with imidazole-containing buffers.

[0066] Also encompassed by the present invention are fusion proteinsthat direct a NHP to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of NHPs to antibody moleculesor their Fab fragments could be used to target cells bearing aparticular epitope. Attaching an appropriate signal sequence to a NHPwould also transport a NHP to a desired location within the cell.Alternatively targeting of a NHP or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes: A Practical Approach”, New,R.R.C., ed., Oxford University Press, N.Y., and in U.S. Pat. Nos.4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respectivedisclosures, which are herein incorporated by reference in theirentirety. Additionally embodied are novel protein constructs engineeredin such a way that they facilitate transport of NHPs to a target site ordesired organ, where they cross the cell membrane and/or the nucleuswhere the NHPs can exert their functional activity. This goal may beachieved by coupling of a NHP to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. Provisional Patent Application Ser. Nos. 60/111,701and 60/056,713, both of which are herein incorporated by reference, forexamples of such transducing sequences), to facilitate passage acrosscellular membranes, and can optionally be engineered to include nuclearlocalization signals.

[0067] Additionally contemplated are oligopeptides that are modeled onan amino acid sequence first described in the Sequence Listing. Such NHPoligopeptides are generally between about 10 to about 100 amino acidslong, or between about 16 to about 80 amino acids long, or between about20 to about 35 amino acids long, or any variation or combination ofsizes represented therein that incorporate a contiguous region ofsequence first disclosed in the Sequence Listing. Such NHP oligopeptidescan be of any length disclosed within the above ranges and can initiateat any amino acid position represented in the Sequence Listing.

[0068] The invention also contemplates “substantially isolated” or“substantially pure” proteins or polypeptides. By a “substantiallyisolated” or “substantially pure” protein or polypeptide is meant aprotein or polypeptide that has been separated from at least some ofthose components that naturally accompany it. Typically, the protein orpolypeptide is substantially isolated or pure when it is at least 60%,by weight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated in vivo. Preferably, thepurity of the preparation is at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight. A substantially isolated orpure protein or polypeptide may be obtained, for example, by extractionfrom a natural source, by expression of a recombinant nucleic acidencoding the protein or polypeptide, or by chemically synthesizing theprotein or polypeptide.

[0069] Purity can be measured by any appropriate method, e.g., columnchromatography such as immunoaffinity chromatography using an antibodyspecific for the protein or polypeptide, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein or polypeptide issubstantially free of naturally associated components when it isseparated from at least some of those contaminants that accompany it inits natural state. Thus, a polypeptide that is chemically synthesized orproduced in a cellular system different from the cell from which itnaturally originates will be, by definition, substantially free from itsnaturally associated components. Accordingly, substantially isolated orpure proteins or polypeptides include eukaryotic proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

5.3 ANTIBODIES TO NHP PRODUCTS

[0070] Antibodies that specifically recognize one or more epitopes of aNHP, epitopes of conserved variants of a NHP, or peptide fragments of aNHP, are also encompassed by the invention. Such antibodies include, butare not limited to, polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0071] The antibodies of the invention may be used, for example, in thedetection of a NHP in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of a NHP. Such antibodiesmay also be utilized in conjunction with, for example, compoundscreening schemes for the evaluation of the effect of test compounds onexpression and/or activity of a NHP expression product. Additionally,such antibodies can be used in conjunction with gene therapy to, forexample, evaluate normal and/or engineered NHP-expressing cells prior totheir introduction into a patient. Such antibodies may additionally beused in methods for the inhibition of abnormal NHP activity. Thus, suchantibodies may be utilized as a part of treatment methods.

[0072] For the production of antibodies, various host animals may beimmunized by injection with a NHP, a NHP peptide (e.g., onecorresponding to a functional domain of a NHP), a truncated NHPpolypeptide (a NHP in which one or more domains have been deleted),functional equivalents of a NHP, or mutated variants of a NHP. Such hostanimals may include, but are not limited to, pigs, rabbits, mice, goats,and rats, to name but a few. Various adjuvants may be used to increasethe immunological response, depending on the host species, including,but not limited to, Freund's adjuvant (complete and incomplete), mineralsalts such as aluminum hydroxide or aluminum phosphate, chitosan,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Alternatively, the immune response could be enhanced bycombination and/or coupling with molecules such as keyhole limpethemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, cholera toxin,or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0073] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof.The hybridomas producing the mAbs of this invention may be cultivated invitro or in vivo. Production of high titers of mAbs in vivo makes thisthe presently preferred method of production.

[0074] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,114,598,6,075,181 and 5,877,397 and their respective disclosures, which areherein incorporated by reference in their entirety. Also encompassed bythe present invention is the use of fully humanized monoclonalantibodies, as described in U.S. Pat. No. 6,150,584 and respectivedisclosures, which are herein incorporated by reference in theirentirety.

[0075] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHP expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0076] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: F(ab′)₂ fragments, which can be produced by pepsindigestion of an antibody molecule; and Fab fragments, which can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0077] Antibodies to a NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniqueswell-known to those skilled in the art (see, e.g., Greenspan and Bona,1993, FASEB J. 7:437-444; and Nissinoff, 1991, J. Immunol.147:2429-2438). For example, antibodies that bind to a NHP domain andcompetitively inhibit the binding of a NHP to its cognate receptor canbe used to generate anti-idiotypes that “mimic” the NHP and, therefore,bind and activate or neutralize a receptor. Such anti-idiotypicantibodies, or Fab fragments of such anti-idiotypes, can be used intherapeutic regimens involving a NHP-mediated pathway.

[0078] Additionally given the high degree of relatedness of mammalianNHPs, NHP knock-out mice (having never seen a NHP, and thus never beentolerized to a NHP) have a unique utility, as they can be advantageouslyapplied to the generation of antibodies against the disclosed mammalianNHPs (i.e., a NHP will be immunogenic in NHP knock-out animals).

[0079] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 8 1 1473 DNA homo sapiens 1 atgcagccag caatgatgat gttttccagtaaatactggg cacggagagg gttttccctg 60 gattcagcag tgcccgaaga gcatcagctacttggcagct caacactaaa taaacctaac 120 tctggcaaaa atgacgacaa aggcaacaagggaagcagca aacgtgaagc tgctaccgaa 180 agtggcaaga cagcagttgt tttctccttgaagaatgaag ttggtggatt ggtaaaagca 240 ctgaggctct ttcaggaaaa acgtgtcaacatggttcata ttgaatccag gaaatctcgg 300 cgaagaagtt ctgaggttga aatctttgtggactgtgagt gtgggaaaac agaattcaat 360 gagctcattc agttgctgaa atttcaaaccactattgtga cgctgaatcc tccagagaac 420 atttggacag aggaagaaga gctagaggatgtgccctggt tccctcggaa gatctctgag 480 ttagacaaat gctctcacag agttctcatgtatggttctg agcttgatgc tgaccaccca 540 ggatttaagg acaatgtcta tcgacagagaagaaagtatt ttgtggatgt ggccatgggt 600 tataaatatg gtcagcccat tcccagggtggagtatactg aagaagaaac taaaacttgg 660 ggtgttgtat tccgggagct ctccaaactctatcccactc atgcttgccg agagtatttg 720 aaaaacttcc ctctgctgac taaatactgtggctacagag aggacaatgt gcctcaactc 780 gaagatgtct ccatgtttct gaaagaaaggtctggcttca cggtgaggcc ggtggctgga 840 tacctgagcc cacgagactt tctggcaggactggcctaca gagtgttcca ctgtacccag 900 tacatccggc atggctcaga tcccctctacaccccagaac cagacacatg ccatgaactc 960 ttgggacatg ttccactact tgcggatcctaagtttgctc agttttcaca agaaataggt 1020 ctggcgtctc tgggagcatc agatgaagatgttcagaaac tagccacgtg ctatttcttc 1080 acaatcgagt ttggcctttg caagcaagaagggcaactgc gggcatatgg agcaggactc 1140 ctttcctcca ttggagaatt aaagcacgccctttctgaca aggcatgtgt gaaagccttt 1200 gacccaaaga caacttgctt acaggaatgccttatcacca ccttccagga agcctacttt 1260 gtttcagaaa gttttgaaga agccaaagaaaagatgaggg actttgcaaa gtcaattacc 1320 cgtcccttct cagtatactt caatccctacacacagagta ttgaaattct gaaagacacc 1380 agaagtattg aaaatgtggt gcaggaccttcgcagcgact tgaatacagt gtgtgatgct 1440 ttaaacaaaa tgaaccaata tctggggatttga 1473 2 490 PRT homo sapiens 2 Met Gln Pro Ala Met Met Met Phe SerSer Lys Tyr Trp Ala Arg Arg 1 5 10 15 Gly Phe Ser Leu Asp Ser Ala ValPro Glu Glu His Gln Leu Leu Gly 20 25 30 Ser Ser Thr Leu Asn Lys Pro AsnSer Gly Lys Asn Asp Asp Lys Gly 35 40 45 Asn Lys Gly Ser Ser Lys Arg GluAla Ala Thr Glu Ser Gly Lys Thr 50 55 60 Ala Val Val Phe Ser Leu Lys AsnGlu Val Gly Gly Leu Val Lys Ala 65 70 75 80 Leu Arg Leu Phe Gln Glu LysArg Val Asn Met Val His Ile Glu Ser 85 90 95 Arg Lys Ser Arg Arg Arg SerSer Glu Val Glu Ile Phe Val Asp Cys 100 105 110 Glu Cys Gly Lys Thr GluPhe Asn Glu Leu Ile Gln Leu Leu Lys Phe 115 120 125 Gln Thr Thr Ile ValThr Leu Asn Pro Pro Glu Asn Ile Trp Thr Glu 130 135 140 Glu Glu Glu LeuGlu Asp Val Pro Trp Phe Pro Arg Lys Ile Ser Glu 145 150 155 160 Leu AspLys Cys Ser His Arg Val Leu Met Tyr Gly Ser Glu Leu Asp 165 170 175 AlaAsp His Pro Gly Phe Lys Asp Asn Val Tyr Arg Gln Arg Arg Lys 180 185 190Tyr Phe Val Asp Val Ala Met Gly Tyr Lys Tyr Gly Gln Pro Ile Pro 195 200205 Arg Val Glu Tyr Thr Glu Glu Glu Thr Lys Thr Trp Gly Val Val Phe 210215 220 Arg Glu Leu Ser Lys Leu Tyr Pro Thr His Ala Cys Arg Glu Tyr Leu225 230 235 240 Lys Asn Phe Pro Leu Leu Thr Lys Tyr Cys Gly Tyr Arg GluAsp Asn 245 250 255 Val Pro Gln Leu Glu Asp Val Ser Met Phe Leu Lys GluArg Ser Gly 260 265 270 Phe Thr Val Arg Pro Val Ala Gly Tyr Leu Ser ProArg Asp Phe Leu 275 280 285 Ala Gly Leu Ala Tyr Arg Val Phe His Cys ThrGln Tyr Ile Arg His 290 295 300 Gly Ser Asp Pro Leu Tyr Thr Pro Glu ProAsp Thr Cys His Glu Leu 305 310 315 320 Leu Gly His Val Pro Leu Leu AlaAsp Pro Lys Phe Ala Gln Phe Ser 325 330 335 Gln Glu Ile Gly Leu Ala SerLeu Gly Ala Ser Asp Glu Asp Val Gln 340 345 350 Lys Leu Ala Thr Cys TyrPhe Phe Thr Ile Glu Phe Gly Leu Cys Lys 355 360 365 Gln Glu Gly Gln LeuArg Ala Tyr Gly Ala Gly Leu Leu Ser Ser Ile 370 375 380 Gly Glu Leu LysHis Ala Leu Ser Asp Lys Ala Cys Val Lys Ala Phe 385 390 395 400 Asp ProLys Thr Thr Cys Leu Gln Glu Cys Leu Ile Thr Thr Phe Gln 405 410 415 GluAla Tyr Phe Val Ser Glu Ser Phe Glu Glu Ala Lys Glu Lys Met 420 425 430Arg Asp Phe Ala Lys Ser Ile Thr Arg Pro Phe Ser Val Tyr Phe Asn 435 440445 Pro Tyr Thr Gln Ser Ile Glu Ile Leu Lys Asp Thr Arg Ser Ile Glu 450455 460 Asn Val Val Gln Asp Leu Arg Ser Asp Leu Asn Thr Val Cys Asp Ala465 470 475 480 Leu Asn Lys Met Asn Gln Tyr Leu Gly Ile 485 490 3 1461DNA homo sapiens 3 atgatgatgt tttccagtaa atactgggca cggagagggttttccctgga ttcagcagtg 60 cccgaagagc atcagctact tggcagctca acactaaataaacctaactc tggcaaaaat 120 gacgacaaag gcaacaaggg aagcagcaaa cgtgaagctgctaccgaaag tggcaagaca 180 gcagttgttt tctccttgaa gaatgaagtt ggtggattggtaaaagcact gaggctcttt 240 caggaaaaac gtgtcaacat ggttcatatt gaatccaggaaatctcggcg aagaagttct 300 gaggttgaaa tctttgtgga ctgtgagtgt gggaaaacagaattcaatga gctcattcag 360 ttgctgaaat ttcaaaccac tattgtgacg ctgaatcctccagagaacat ttggacagag 420 gaagaagagc tagaggatgt gccctggttc cctcggaagatctctgagtt agacaaatgc 480 tctcacagag ttctcatgta tggttctgag cttgatgctgaccacccagg atttaaggac 540 aatgtctatc gacagagaag aaagtatttt gtggatgtggccatgggtta taaatatggt 600 cagcccattc ccagggtgga gtatactgaa gaagaaactaaaacttgggg tgttgtattc 660 cgggagctct ccaaactcta tcccactcat gcttgccgagagtatttgaa aaacttccct 720 ctgctgacta aatactgtgg ctacagagag gacaatgtgcctcaactcga agatgtctcc 780 atgtttctga aagaaaggtc tggcttcacg gtgaggccggtggctggata cctgagccca 840 cgagactttc tggcaggact ggcctacaga gtgttccactgtacccagta catccggcat 900 ggctcagatc ccctctacac cccagaacca gacacatgccatgaactctt gggacatgtt 960 ccactacttg cggatcctaa gtttgctcag ttttcacaagaaataggtct ggcgtctctg 1020 ggagcatcag atgaagatgt tcagaaacta gccacgtgctatttcttcac aatcgagttt 1080 ggcctttgca agcaagaagg gcaactgcgg gcatatggagcaggactcct ttcctccatt 1140 ggagaattaa agcacgccct ttctgacaag gcatgtgtgaaagcctttga cccaaagaca 1200 acttgcttac aggaatgcct tatcaccacc ttccaggaagcctactttgt ttcagaaagt 1260 tttgaagaag ccaaagaaaa gatgagggac tttgcaaagtcaattacccg tcccttctca 1320 gtatacttca atccctacac acagagtatt gaaattctgaaagacaccag aagtattgaa 1380 aatgtggtgc aggaccttcg cagcgacttg aatacagtgtgtgatgcttt aaacaaaatg 1440 aaccaatatc tggggatttg a 1461 4 486 PRT homosapiens 4 Met Met Met Phe Ser Ser Lys Tyr Trp Ala Arg Arg Gly Phe SerLeu 1 5 10 15 Asp Ser Ala Val Pro Glu Glu His Gln Leu Leu Gly Ser SerThr Leu 20 25 30 Asn Lys Pro Asn Ser Gly Lys Asn Asp Asp Lys Gly Asn LysGly Ser 35 40 45 Ser Lys Arg Glu Ala Ala Thr Glu Ser Gly Lys Thr Ala ValVal Phe 50 55 60 Ser Leu Lys Asn Glu Val Gly Gly Leu Val Lys Ala Leu ArgLeu Phe 65 70 75 80 Gln Glu Lys Arg Val Asn Met Val His Ile Glu Ser ArgLys Ser Arg 85 90 95 Arg Arg Ser Ser Glu Val Glu Ile Phe Val Asp Cys GluCys Gly Lys 100 105 110 Thr Glu Phe Asn Glu Leu Ile Gln Leu Leu Lys PheGln Thr Thr Ile 115 120 125 Val Thr Leu Asn Pro Pro Glu Asn Ile Trp ThrGlu Glu Glu Glu Leu 130 135 140 Glu Asp Val Pro Trp Phe Pro Arg Lys IleSer Glu Leu Asp Lys Cys 145 150 155 160 Ser His Arg Val Leu Met Tyr GlySer Glu Leu Asp Ala Asp His Pro 165 170 175 Gly Phe Lys Asp Asn Val TyrArg Gln Arg Arg Lys Tyr Phe Val Asp 180 185 190 Val Ala Met Gly Tyr LysTyr Gly Gln Pro Ile Pro Arg Val Glu Tyr 195 200 205 Thr Glu Glu Glu ThrLys Thr Trp Gly Val Val Phe Arg Glu Leu Ser 210 215 220 Lys Leu Tyr ProThr His Ala Cys Arg Glu Tyr Leu Lys Asn Phe Pro 225 230 235 240 Leu LeuThr Lys Tyr Cys Gly Tyr Arg Glu Asp Asn Val Pro Gln Leu 245 250 255 GluAsp Val Ser Met Phe Leu Lys Glu Arg Ser Gly Phe Thr Val Arg 260 265 270Pro Val Ala Gly Tyr Leu Ser Pro Arg Asp Phe Leu Ala Gly Leu Ala 275 280285 Tyr Arg Val Phe His Cys Thr Gln Tyr Ile Arg His Gly Ser Asp Pro 290295 300 Leu Tyr Thr Pro Glu Pro Asp Thr Cys His Glu Leu Leu Gly His Val305 310 315 320 Pro Leu Leu Ala Asp Pro Lys Phe Ala Gln Phe Ser Gln GluIle Gly 325 330 335 Leu Ala Ser Leu Gly Ala Ser Asp Glu Asp Val Gln LysLeu Ala Thr 340 345 350 Cys Tyr Phe Phe Thr Ile Glu Phe Gly Leu Cys LysGln Glu Gly Gln 355 360 365 Leu Arg Ala Tyr Gly Ala Gly Leu Leu Ser SerIle Gly Glu Leu Lys 370 375 380 His Ala Leu Ser Asp Lys Ala Cys Val LysAla Phe Asp Pro Lys Thr 385 390 395 400 Thr Cys Leu Gln Glu Cys Leu IleThr Thr Phe Gln Glu Ala Tyr Phe 405 410 415 Val Ser Glu Ser Phe Glu GluAla Lys Glu Lys Met Arg Asp Phe Ala 420 425 430 Lys Ser Ile Thr Arg ProPhe Ser Val Tyr Phe Asn Pro Tyr Thr Gln 435 440 445 Ser Ile Glu Ile LeuLys Asp Thr Arg Ser Ile Glu Asn Val Val Gln 450 455 460 Asp Leu Arg SerAsp Leu Asn Thr Val Cys Asp Ala Leu Asn Lys Met 465 470 475 480 Asn GlnTyr Leu Gly Ile 485 5 1458 DNA homo sapiens 5 atgatgtttt ccagtaaatactgggcacgg agagggtttt ccctggattc agcagtgccc 60 gaagagcatc agctacttggcagctcaaca ctaaataaac ctaactctgg caaaaatgac 120 gacaaaggca acaagggaagcagcaaacgt gaagctgcta ccgaaagtgg caagacagca 180 gttgttttct ccttgaagaatgaagttggt ggattggtaa aagcactgag gctctttcag 240 gaaaaacgtg tcaacatggttcatattgaa tccaggaaat ctcggcgaag aagttctgag 300 gttgaaatct ttgtggactgtgagtgtggg aaaacagaat tcaatgagct cattcagttg 360 ctgaaatttc aaaccactattgtgacgctg aatcctccag agaacatttg gacagaggaa 420 gaagagctag aggatgtgccctggttccct cggaagatct ctgagttaga caaatgctct 480 cacagagttc tcatgtatggttctgagctt gatgctgacc acccaggatt taaggacaat 540 gtctatcgac agagaagaaagtattttgtg gatgtggcca tgggttataa atatggtcag 600 cccattccca gggtggagtatactgaagaa gaaactaaaa cttggggtgt tgtattccgg 660 gagctctcca aactctatcccactcatgct tgccgagagt atttgaaaaa cttccctctg 720 ctgactaaat actgtggctacagagaggac aatgtgcctc aactcgaaga tgtctccatg 780 tttctgaaag aaaggtctggcttcacggtg aggccggtgg ctggatacct gagcccacga 840 gactttctgg caggactggcctacagagtg ttccactgta cccagtacat ccggcatggc 900 tcagatcccc tctacaccccagaaccagac acatgccatg aactcttggg acatgttcca 960 ctacttgcgg atcctaagtttgctcagttt tcacaagaaa taggtctggc gtctctggga 1020 gcatcagatg aagatgttcagaaactagcc acgtgctatt tcttcacaat cgagtttggc 1080 ctttgcaagc aagaagggcaactgcgggca tatggagcag gactcctttc ctccattgga 1140 gaattaaagc acgccctttctgacaaggca tgtgtgaaag cctttgaccc aaagacaact 1200 tgcttacagg aatgccttatcaccaccttc caggaagcct actttgtttc agaaagtttt 1260 gaagaagcca aagaaaagatgagggacttt gcaaagtcaa ttacccgtcc cttctcagta 1320 tacttcaatc cctacacacagagtattgaa attctgaaag acaccagaag tattgaaaat 1380 gtggtgcagg accttcgcagcgacttgaat acagtgtgtg atgctttaaa caaaatgaac 1440 caatatctgg ggatttga1458 6 485 PRT homo sapiens 6 Met Met Phe Ser Ser Lys Tyr Trp Ala ArgArg Gly Phe Ser Leu Asp 1 5 10 15 Ser Ala Val Pro Glu Glu His Gln LeuLeu Gly Ser Ser Thr Leu Asn 20 25 30 Lys Pro Asn Ser Gly Lys Asn Asp AspLys Gly Asn Lys Gly Ser Ser 35 40 45 Lys Arg Glu Ala Ala Thr Glu Ser GlyLys Thr Ala Val Val Phe Ser 50 55 60 Leu Lys Asn Glu Val Gly Gly Leu ValLys Ala Leu Arg Leu Phe Gln 65 70 75 80 Glu Lys Arg Val Asn Met Val HisIle Glu Ser Arg Lys Ser Arg Arg 85 90 95 Arg Ser Ser Glu Val Glu Ile PheVal Asp Cys Glu Cys Gly Lys Thr 100 105 110 Glu Phe Asn Glu Leu Ile GlnLeu Leu Lys Phe Gln Thr Thr Ile Val 115 120 125 Thr Leu Asn Pro Pro GluAsn Ile Trp Thr Glu Glu Glu Glu Leu Glu 130 135 140 Asp Val Pro Trp PhePro Arg Lys Ile Ser Glu Leu Asp Lys Cys Ser 145 150 155 160 His Arg ValLeu Met Tyr Gly Ser Glu Leu Asp Ala Asp His Pro Gly 165 170 175 Phe LysAsp Asn Val Tyr Arg Gln Arg Arg Lys Tyr Phe Val Asp Val 180 185 190 AlaMet Gly Tyr Lys Tyr Gly Gln Pro Ile Pro Arg Val Glu Tyr Thr 195 200 205Glu Glu Glu Thr Lys Thr Trp Gly Val Val Phe Arg Glu Leu Ser Lys 210 215220 Leu Tyr Pro Thr His Ala Cys Arg Glu Tyr Leu Lys Asn Phe Pro Leu 225230 235 240 Leu Thr Lys Tyr Cys Gly Tyr Arg Glu Asp Asn Val Pro Gln LeuGlu 245 250 255 Asp Val Ser Met Phe Leu Lys Glu Arg Ser Gly Phe Thr ValArg Pro 260 265 270 Val Ala Gly Tyr Leu Ser Pro Arg Asp Phe Leu Ala GlyLeu Ala Tyr 275 280 285 Arg Val Phe His Cys Thr Gln Tyr Ile Arg His GlySer Asp Pro Leu 290 295 300 Tyr Thr Pro Glu Pro Asp Thr Cys His Glu LeuLeu Gly His Val Pro 305 310 315 320 Leu Leu Ala Asp Pro Lys Phe Ala GlnPhe Ser Gln Glu Ile Gly Leu 325 330 335 Ala Ser Leu Gly Ala Ser Asp GluAsp Val Gln Lys Leu Ala Thr Cys 340 345 350 Tyr Phe Phe Thr Ile Glu PheGly Leu Cys Lys Gln Glu Gly Gln Leu 355 360 365 Arg Ala Tyr Gly Ala GlyLeu Leu Ser Ser Ile Gly Glu Leu Lys His 370 375 380 Ala Leu Ser Asp LysAla Cys Val Lys Ala Phe Asp Pro Lys Thr Thr 385 390 395 400 Cys Leu GlnGlu Cys Leu Ile Thr Thr Phe Gln Glu Ala Tyr Phe Val 405 410 415 Ser GluSer Phe Glu Glu Ala Lys Glu Lys Met Arg Asp Phe Ala Lys 420 425 430 SerIle Thr Arg Pro Phe Ser Val Tyr Phe Asn Pro Tyr Thr Gln Ser 435 440 445Ile Glu Ile Leu Lys Asp Thr Arg Ser Ile Glu Asn Val Val Gln Asp 450 455460 Leu Arg Ser Asp Leu Asn Thr Val Cys Asp Ala Leu Asn Lys Met Asn 465470 475 480 Gln Tyr Leu Gly Ile 485 7 1455 DNA homo sapiens 7 atgttttccagtaaatactg ggcacggaga gggttttccc tggattcagc agtgcccgaa 60 gagcatcagctacttggcag ctcaacacta aataaaccta actctggcaa aaatgacgac 120 aaaggcaacaagggaagcag caaacgtgaa gctgctaccg aaagtggcaa gacagcagtt 180 gttttctccttgaagaatga agttggtgga ttggtaaaag cactgaggct ctttcaggaa 240 aaacgtgtcaacatggttca tattgaatcc aggaaatctc ggcgaagaag ttctgaggtt 300 gaaatctttgtggactgtga gtgtgggaaa acagaattca atgagctcat tcagttgctg 360 aaatttcaaaccactattgt gacgctgaat cctccagaga acatttggac agaggaagaa 420 gagctagaggatgtgccctg gttccctcgg aagatctctg agttagacaa atgctctcac 480 agagttctcatgtatggttc tgagcttgat gctgaccacc caggatttaa ggacaatgtc 540 tatcgacagagaagaaagta ttttgtggat gtggccatgg gttataaata tggtcagccc 600 attcccagggtggagtatac tgaagaagaa actaaaactt ggggtgttgt attccgggag 660 ctctccaaactctatcccac tcatgcttgc cgagagtatt tgaaaaactt ccctctgctg 720 actaaatactgtggctacag agaggacaat gtgcctcaac tcgaagatgt ctccatgttt 780 ctgaaagaaaggtctggctt cacggtgagg ccggtggctg gatacctgag cccacgagac 840 tttctggcaggactggccta cagagtgttc cactgtaccc agtacatccg gcatggctca 900 gatcccctctacaccccaga accagacaca tgccatgaac tcttgggaca tgttccacta 960 cttgcggatcctaagtttgc tcagttttca caagaaatag gtctggcgtc tctgggagca 1020 tcagatgaagatgttcagaa actagccacg tgctatttct tcacaatcga gtttggcctt 1080 tgcaagcaagaagggcaact gcgggcatat ggagcaggac tcctttcctc cattggagaa 1140 ttaaagcacgccctttctga caaggcatgt gtgaaagcct ttgacccaaa gacaacttgc 1200 ttacaggaatgccttatcac caccttccag gaagcctact ttgtttcaga aagttttgaa 1260 gaagccaaagaaaagatgag ggactttgca aagtcaatta cccgtccctt ctcagtatac 1320 ttcaatccctacacacagag tattgaaatt ctgaaagaca ccagaagtat tgaaaatgtg 1380 gtgcaggaccttcgcagcga cttgaataca gtgtgtgatg ctttaaacaa aatgaaccaa 1440 tatctggggatttga 1455 8 484 PRT homo sapiens 8 Met Phe Ser Ser Lys Tyr Trp Ala ArgArg Gly Phe Ser Leu Asp Ser 1 5 10 15 Ala Val Pro Glu Glu His Gln LeuLeu Gly Ser Ser Thr Leu Asn Lys 20 25 30 Pro Asn Ser Gly Lys Asn Asp AspLys Gly Asn Lys Gly Ser Ser Lys 35 40 45 Arg Glu Ala Ala Thr Glu Ser GlyLys Thr Ala Val Val Phe Ser Leu 50 55 60 Lys Asn Glu Val Gly Gly Leu ValLys Ala Leu Arg Leu Phe Gln Glu 65 70 75 80 Lys Arg Val Asn Met Val HisIle Glu Ser Arg Lys Ser Arg Arg Arg 85 90 95 Ser Ser Glu Val Glu Ile PheVal Asp Cys Glu Cys Gly Lys Thr Glu 100 105 110 Phe Asn Glu Leu Ile GlnLeu Leu Lys Phe Gln Thr Thr Ile Val Thr 115 120 125 Leu Asn Pro Pro GluAsn Ile Trp Thr Glu Glu Glu Glu Leu Glu Asp 130 135 140 Val Pro Trp PhePro Arg Lys Ile Ser Glu Leu Asp Lys Cys Ser His 145 150 155 160 Arg ValLeu Met Tyr Gly Ser Glu Leu Asp Ala Asp His Pro Gly Phe 165 170 175 LysAsp Asn Val Tyr Arg Gln Arg Arg Lys Tyr Phe Val Asp Val Ala 180 185 190Met Gly Tyr Lys Tyr Gly Gln Pro Ile Pro Arg Val Glu Tyr Thr Glu 195 200205 Glu Glu Thr Lys Thr Trp Gly Val Val Phe Arg Glu Leu Ser Lys Leu 210215 220 Tyr Pro Thr His Ala Cys Arg Glu Tyr Leu Lys Asn Phe Pro Leu Leu225 230 235 240 Thr Lys Tyr Cys Gly Tyr Arg Glu Asp Asn Val Pro Gln LeuGlu Asp 245 250 255 Val Ser Met Phe Leu Lys Glu Arg Ser Gly Phe Thr ValArg Pro Val 260 265 270 Ala Gly Tyr Leu Ser Pro Arg Asp Phe Leu Ala GlyLeu Ala Tyr Arg 275 280 285 Val Phe His Cys Thr Gln Tyr Ile Arg His GlySer Asp Pro Leu Tyr 290 295 300 Thr Pro Glu Pro Asp Thr Cys His Glu LeuLeu Gly His Val Pro Leu 305 310 315 320 Leu Ala Asp Pro Lys Phe Ala GlnPhe Ser Gln Glu Ile Gly Leu Ala 325 330 335 Ser Leu Gly Ala Ser Asp GluAsp Val Gln Lys Leu Ala Thr Cys Tyr 340 345 350 Phe Phe Thr Ile Glu PheGly Leu Cys Lys Gln Glu Gly Gln Leu Arg 355 360 365 Ala Tyr Gly Ala GlyLeu Leu Ser Ser Ile Gly Glu Leu Lys His Ala 370 375 380 Leu Ser Asp LysAla Cys Val Lys Ala Phe Asp Pro Lys Thr Thr Cys 385 390 395 400 Leu GlnGlu Cys Leu Ile Thr Thr Phe Gln Glu Ala Tyr Phe Val Ser 405 410 415 GluSer Phe Glu Glu Ala Lys Glu Lys Met Arg Asp Phe Ala Lys Ser 420 425 430Ile Thr Arg Pro Phe Ser Val Tyr Phe Asn Pro Tyr Thr Gln Ser Ile 435 440445 Glu Ile Leu Lys Asp Thr Arg Ser Ile Glu Asn Val Val Gln Asp Leu 450455 460 Arg Ser Asp Leu Asn Thr Val Cys Asp Ala Leu Asn Lys Met Asn Gln465 470 475 480 Tyr Leu Gly Ile

What is claimed is:
 1. An isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:7.
 2. The isolated nucleic acidmolecule of claim 1, wherein the molecule comprises the nucleotidesequence of SEQ ID NO:5.
 3. The isolated nucleic acid molecule of claim2, wherein the molecule comprises the nucleotide sequence of SEQ IDNO:3.
 4. The isolated nucleic acid molecule of claim 3, wherein themolecule comprises the nucleotide sequence of SEQ ID NO:1.
 5. Anisolated nucleic acid molecule comprising a nucleotide sequence that:(a) encodes the amino acid sequence shown in SEQ ID NO:4; and (b)hybridizes under highly stringent conditions to the nucleotide sequenceof SEQ ID NO:3 or the complement thereof.
 6. An isolated nucleic acidmolecule encoding the amino acid sequence described in SEQ ID NO:8. 7.The isolated nucleic acid molecule of claim 6, wherein the moleculeencodes the amino acid sequence of SEQ ID NO:
 6. 8. The isolated nucleicacid molecule of claim 7, wherein the molecule encodes the amino acidsequence of SEQ ID NO:4.
 9. The isolated nucleic acid molecule of claim8, wherein the molecule encodes the amino acid sequence of SEQ ID NO:2.10. A substantially isolated protein having the activity of the proteinshown in SEQ ID NOS:2, 4, 6, or 8, which is encoded by a nucleotidesequence that hybridizes to SEQ ID NO:1, 3, 5, or 7 under highlystringent conditions.